臺灣博碩士論文加值系統

犬乳腺腫瘤是母犬最常發生的腫瘤之一，而單純型乳腺癌 (simple carcinoma)及複合型乳腺癌 (complex carcinoma) 為惡性犬乳腺腫瘤中最主要的兩種類型。為了尋找犬乳腺癌的蛋白質生物標記，本實驗室先前針對乳腺腫瘤組織進行蛋白質體學分析，已證實thymidylate synthase (TYMS)在犬乳腺癌腫瘤組織中的表現量高於正常或是增生的乳腺組織。TYMS是生物體內重要的酵素，能將去氧尿苷單磷酸(Deoxyuridine monophosphated, dUMP)催化成去氧胸苷單磷酸(Deoxythymidine monophosphate, dTMP)參與DNA之合成及修復。本研究進一步以免疫組織化學法驗證TYMS在犬乳腺癌腫瘤組織中之過量表現。已知腫瘤相關抗原可能會誘發動物體的免疫反應，進而產生該抗原的自體抗體。近年有研究指出，在人類乳腺腫瘤病患血清中，可偵測到抗TYMS之自體抗體，有潛力作為偵測乳腺腫瘤的生物標記。因此本研究表現犬TYMS重組蛋白質作為抗原，建立酵素連結免疫分析法 (ELISA)以偵測乳腺腫瘤病犬血清中抗TYMS自體抗體的含量。分析的血清樣本來自於本校獸醫教學醫院69隻乳腺腫瘤病犬及26隻健康母犬，乳腺腫瘤病犬包含20隻單純型乳腺癌、23隻複合型乳腺癌及10隻多重惡性腫瘤病犬 (包含2種或2種以上的惡性腫瘤)。結果顯示，乳腺癌病犬及健康母犬血清中抗TYMS自體抗體的含量於統計學上無顯著差異，然而進一步比較年齡大於等於8歲之所有乳腺癌病犬 (48隻)及健康母犬 (7隻)時，其血清中抗TYMS自體抗體的含量顯著高於健康母犬(p < 0.01)；依照腫瘤類型區分後發現，單純型乳腺癌病犬 (17隻)、複合型乳腺癌病犬 (21隻)、或多重惡性腫瘤病犬 (10隻)，其血清中抗TYMS自體抗體的含量均顯著高於健康母犬 (p < 0.05)。此外，整體臨床分期早期的乳腺腫瘤病犬 (36隻)，其血清中抗TYMS自體抗體的含量顯著高於健康母犬 (26隻，p < 0.05)，而進一步比較其中8歲齡(含)以上之病犬 (31隻)與健康母犬 (7隻)，其差異更為顯著 (p < 0.01)。綜合來說，本研究是第一個偵測犬乳腺腫瘤病犬血清中抗TYMS自體抗體的研究，因為臨床分期早期的乳腺腫瘤病犬血清中抗TYMS自體抗體的含量顯著上升，因此有潛力作為犬乳腺腫瘤早期診斷之生物標記。

Canine mammary gland tumors (CMTs) are the most common tumor in female canine, of which simple carcinoma and complex carcinoma are the two major malignant types. Our lab has analyzed the tissue proteomes of canine CMT tissue, and identified that thymidylate synthase was particularly over-expressed in CMT tissues. TYMS is an essential enzyme that catalyses the transfer of a methyl group from methylenetetrahydrofolate to dUMP to generate dTMP and hence contributes to DNA synthesis and repair. The elevated protein level of TYMS in CMT tissues has been previously verified by Western blot and further by immunohistochemistry in this study. Tumor-associated antigens (TAAs) may induce the immune response of animals in the forms of autoantibodies (AAbs) against the TAAs. It has been recently reported that AAbs against TYMS are elicited in sera of human breast cancer patients, which has a potential to be used as biomarkers for detection of cancer. In this study, the presence of TYMS AAbs in sera of CMT dogs were assessed by establishing an ELISA using recombinant canine TYMS proteins as antigens. Serum samples collected from 69 female CMT dogs and 26 female healthy donors. The dogs with CMT consisted of 20 cases with simple carcinoma, 23 cases with complex carcinoma, and 10 cases with multiple cancers, which comprise more than one type of cancer. The data revealed that there was no significant difference in the levels of seral TYMS AAbs between CMT dogs and healthy donors. Nevertheless, when further comparing the dogs with age ≥ 8 yr, there was statistical significance in comparison of the levels of seral TYMS AAbs between cancer dogs (n = 48) and healthy dogs (p < 0.01), dogs with simple carcinoma (n = 17) and healthy dogs (p < 0.05), dogs with complex carcinoma (n = 21) and healthy dogs (p < 0.05), and dogs with multiple cancer (n = 10) and healthy dogs (p < 0.05). Moreover, the levels of seral TYMS AAbs were significantly elevated in the early-stage caner group (n = 36) compared with the healthy group (p < 0.05), and the significance was enhanced when comparing the dogs older than 8 yr between groups. Taken together, this study reveals for the first time that TYMS AAbs are detectable in canine sera, and the elevated levels of TYMS AAbs may be associated with CMT in early stage, which may be a potential protein biomarker of early detection of CMT.

摘要 i
Abstract ii
目錄 iv
縮寫表 v
圖次 vi
表次 viii
第一章、 背景介紹 1
第一節、犬乳腺腫瘤簡介 1
第二節、生物標記 (biomarker) 4
第三節、胸腺嘧啶合成酶 (thymidylate synthase, TYMS) 6
第四節、自體抗體 7
第二章、 材料與方法 9
第一節、乳腺腫瘤病例血清收集及倫理聲明 9
第二節、SDS-PAGE及coomassie blue染色 9
第三節、西方墨點法 (Western blotting) 10
第四節、電泳分析 11
第五節、構築pET-24a(+)-canine TYMS 11
第六節、在E.coli BL21(DE3)中表現His-tagged canine TYMS蛋白質 12
第七節、純化His-tagged canine TYMS重組蛋白質 13
第八節、蛋白質定量 14
第九節、酵素連結免疫吸附分析法 (ELISA) 14
第十節、統計分析 15
第十一節、免疫組織化學染色 (Immunohistochemistry, IHC) 15
第三章、 結果 16
第一節、免疫組織化學染色 (Immunohistochemistry, IHC) 16
第二節、構築pET-24a(+)-canine TYMS表現載體 16
第三節、在E.coli BL21(DE3)中表現His-tagged canine TYMS重組蛋白質 17
第四節、純化His-tagged canine TYMS重組蛋白質 17
第五節、酵素連結免疫吸附分析法 (ELISA) 18
第四章、 討論 22
第五章、 參考文獻 58

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